Project Summary/Abstract: Penicillin binding protein 2a (PBP2a) is the leading cause of ?-lactam antibiotic resistance in deadly Staphylococcus aureus and Staphylococcus epidermidis infections. Morbidity, mortality, and health care costs create a critical need for antibiotics that can overcome PBP2a. New antibiotic development has led to success against planktonic bacteria but methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE) biofilms continue to cause deadly hospital-acquired infections (HAIs). These factors are barriers to the ?prompt and serious action? urged by the Centers for Disease Control. As discovered in our laboratory, ?-lactam antibiotics that kill methicillin-susceptible S. aureus also prevent the growth of methicillin-resistant S. aureus (MRSA) if administered with branched poly(ethylenimine), BPEI. The ?-lactam + BPEI combinations are also effective against exopolymers surround MRSE bacteria. This route to reduce morbidity, mortality, and health care costs will remain closed without experiments to maximize potency. Our long-term goal is to kill bacterial pathogens and their associated biofilms. The overall objective is to determine if antibiotics that target bacterial pathogens (?-lactams, vancomycin, linezolid, rifampicin) are potentiated against MRSA and MRSE that express biofilm extracellular polymeric substances (EPS) and the mecA gene responsible for PBP2a expression. The central hypothesis is that resistance from EPS and PBP2a can be conquered when wall teichoic acid (WTA) is disabled by cationic polymer potentiators. This effect may arise from electrostatic interactions between BPEI and WTA. The rationale underlying the proposed research is that BPEI disrupts the biofilm architecture and counteracts resistance from mecA, making MRSA and MRSE susceptible to ?-lactam antibiotics. In our opinion, the rationale departs from the status quo of stopping WTA biosynthesis. Low protein binding and potency in serum are retained. Drug safety is increased by linking potentiators to non-toxic poly(ethylene glycol), PEG, molecules. An in vivo study shows the maximum tolerable dose is over 200 mg/kg. The study design to test the central hypothesis involves pursuit of the following specific aims. Aim 1, Create a library of anti-biofilm potentiators. Aim 2, Identify which potentiators have greatest anti-biofilm activity when used in combination with antibiotics. Data from these aims will demonstrate the possibility of improving the health outcomes of persons afflicted with staphylococcal infections. In our opinion, this route is innovative by the use of PEGylated potentiators to deactivate anionic teichoic acid through electrostatic interactions. The impact on the research community is a pathway that enables vertical advancement of antibiotic drug discovery by providing ways for other researchers to reinvigorate efforts that have failed to overcome resistance in biofilms.